Herpes simplex virus (HSV) remains a major human pathogen world-wide causing human diseases including cold sores, ocular and genital infections, neonatal infections and encephalitis. HSV establishes a life-long latent infection which reactivates periodically to produce recurrent disease. The long-term objective of this research is to define the mechanism of HSV entry into host cells. Understanding of the initial events in HSV infection may help develop new treatments and aid with viral targeting approaches for gene therapy protocols. HSV has long been recognized to enter some cells by pH-neutral fusion with the plasma membrane. Recently, we found that initial infection of some common cultured cell types requires endocytosis and low intracellular pH. Thus, HSV uses more than one pathway to deliver its genome to the cell interior. HSV infects many diverse cultured cell types and has multiple targets in vivo including cells of epithelial and neuronal origin. This relatively broad host range may be explained by multiple entry pathways. The process of HSV entry via endocytosis includes two sequential stages: internalization of intact, enveloped virus from the cell surface, and penetration of the capsid into the cytosol. I propose that these two stages are separable and have distinct molecular requirements. The specific aims of the proposed research are: (1) to characterize the cellular mechanism of HSV entry into human epithelial cells; and (2) to identify the viral requirements of HSV entry via endocytosis. A combination of molecular, biochemical and cell biological approaches will be used to achieve these goals. The results will define the determinants for entry of HSV into relevant human cell types. Additionally, these studies should elucidate the mechanisms that target the incoming virus to the proper cellular compartments to initiate productive infection. [unreadable] [unreadable]